CN101718794A - Preparation method of ABO/RhD blood typing detection reagent card - Google Patents
Preparation method of ABO/RhD blood typing detection reagent card Download PDFInfo
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- CN101718794A CN101718794A CN 200910234608 CN200910234608A CN101718794A CN 101718794 A CN101718794 A CN 101718794A CN 200910234608 CN200910234608 CN 200910234608 CN 200910234608 A CN200910234608 A CN 200910234608A CN 101718794 A CN101718794 A CN 101718794A
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Abstract
The invention relates to a preparation method of an ABO/RhD blood typing detection reagent card. Six microcolumn gel tubes are arranged on the reagent card and includes a gel tube containing monoclonal anti-A antibodies with IgM property, a gel tube containing monoclonal anti-B antibodies with IgM property, a gel tube containing monoclonal anti-D antibodies with IgM property, two gel tubes which are used for reverse typing and contain a gel suspending medium and a gel tube which is used as a negative control and contains the gel suspending medium. The preparation method comprises the processes of preparing the gel suspending medium, preparing gel, selecting antibodies as well as suspending and packing the gel. The ABO/RhD blood typing detection reagent card prepared by the method has high sensitivity, good specificity and stable quality and can be used for positive typing and reverse typing.
Description
(1) technical field
The present invention relates to a kind of blood typing card and application thereof.In particular, the present invention relates to a kind of composition, preparation method and application thereof of ABO/RhD blood typing detection reagent card, relate to the blood transfusion medical domain.
(2) background technology
Since 1900 find abo blood group, at first proposed blood group by Hoktoen (U.S. scientist Hai Kedun) and identified importance in treatment of blood transfusion in 1907.Other scholars had proposed to intersect the notion that cooperates experiment again in 1908, had emphasized the importance that blood group is identified.Blood transfusion is one of important medical procedure of clinical rescue patient life, identifies that correctly blood group is the first step of safe transfusion, and abo blood group is incompatible can to cause the serious i.e. property sent out hemolytic blood transfusion reaction in clinical blood transfusion, jeopardize patient's life.Therefore, the accurate typing of abo blood group is most important, is the basis and the guarantee of safe transfusion.
The Rh blood group system is the important blood group system that is only second to ABO blood group system, also is the most complicated in an erythrocyte blood type system system, and present international Blood Transfusion Association (ISBT) has confirmed that the Rh blood group antigens have 48.5 kinds of major antigens of Rh blood group system are D, C, E, c, e antigen, and wherein D antigen antigenicity is the strongest.It is the RhD positive that erythrocyte surface contains D antigen, and it is the RhD feminine gender that erythrocyte surface does not contain D antigen.After the RhD negative patient accepts the RhD positive blood, have at least 20% patient after contacting this antigen in the body second time, hemolytic reaction can take place to D antigen generation immune response.In addition, when the negative gravid woman's of RhD fetus was the RhD positive, when childbirth or miscarriage, fetal erythrocyte may enter parent by placenta, causes parent to D antigen generation immune response, when this women is pregnant once more, neonatal hemolytic disease may take place.
Ministry of Public Health's requirement of just sending the documents in 2000, stipulate every blood supply and be subjected to the individuality of blood all will carry out ABO, (Ministry of Public Health defends the doctor and sends out (2000) No. 184 files) identified in the Rh blood typing.
At home and abroad, there has been the part manufacturer production to be used for the ABO/RhD blood typing detection reagent card of abo blood group typing and RhD typing, but its gel that adopts is a sephadex, the grain size of this gel is more than 70 nanometers, particle is bigger, slit between the gel is bigger, thereby makes the sensitivity of carrying out the abo blood group typing reduce.In addition, existing card only carries out the ABO positive definite form, does not carry out the ABO reverse type, has reduced the accuracy of abo blood group typing, causes the mistake of abo blood group typing easily.
(3) summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, provide a kind of highly sensitive, specificity good, steady quality, can carry out the preparation method of the ABO/RhD blood typing detection reagent card of positive reverse type simultaneously.
The object of the present invention is achieved like this: a kind of preparation method of ABO/RhD blood typing detection reagent card, have 6 micro-column gel pipes on the described reagent card, it is respectively the gel tube that a pipe contains the monoclonal anti-A antibody of IgM character, one pipe contains the gel tube of the monoclonal anti-B antibody of IgM character, one pipe contains the gel tube of the monoclonal anti-D of IgM character, two pipes are used for the gel tube that contains the gel suspending medium of reverse type and the gel tube that contains the gel suspending medium that a pipe is used for negative control
Described method comprises following technological process:
The preparation of step 1, gel suspending medium
Described gel suspending medium prescription is as follows:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤4%.
Above reagent dissolved in distilled water, adjust pH are 6.6-6.8.
The preparation of step 2, gel
Select the Sephacryl gel for use, grain size is a rice in the 30-60.Wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation, remove the gel particle of damaged fragment of gel and gathering, collect and obtain even particle size and the complete gel that is suitable for spherical in shape.
The selection of step 3, antibody
Select the monoclonal anti-A antibody of IgM character, tire 〉=128
Select the monoclonal anti-B antibody of IgM character, tire 〉=128
Select the monoclonal anti-D of IgM character, tire 〉=64.
The preparation of step 4, gel
The gel of step 2 preparation is mixed according to 2: 1～6: 1 ratio of volume ratio with each antibody of step 3 selection, be mixed with respectively the monoclonal anti-A antibody that contains IgM character gel, contain IgM character the monoclonal anti-B antibody gel and contain the gel of the monoclonal anti-D of IgM character; The gel of step 2 preparation is mixed according to 2: 1～6: 1 ratio of volume ratio with the gel suspending medium of step 1 preparation, be mixed with the gel that contains the gel suspending medium.
Step 5, packing
According to the amount of every pipe 22～28 microlitres, each gel that step 4 is prepared joins respectively in six microtrabeculae pipes of a blank card, forms the ABO/RhD blood typing detection reagent card with 6 micro-column gel pipes.
The invention has the beneficial effects as follows:
First aspect of the present invention has proposed a kind of standardized gel suspending medium system, is used for the washing and the suspension of gel, and can keep the stable of antibody and gel for a long time, and its characteristics are:
1, designed the buffer system with very strong surge capability, the buffer system that adopts potassium phosphate, sodium salt and amino acid to form makes the pH value of system maintain 6.6-6.8.Compare with the single citric acid buffer system that adopt usually in the blood transfusion field, have the stronger characteristics of surge capability, help keeping whole system to be in the buffering range of requirement, guaranteed the ionic strength of whole gel suspending medium simultaneously.
2, has more effectively low salt concn system, form as can be seen from it, gel suspending medium of the present invention adopts amino acid and sodium chloride as adjuvant, assist and keep the low ionic strength environment with the phosphate of trace, keep gel particle to be ball particle and abundant swelling, and the diameter of keeping gel particle is in desired scope.
3, the present invention has unique lubricating system, adopt certain density bovine protein, methyl p-hydroxybenzoate and propylparaben, make and obtain the proper lubrication ability when red blood cell passes through the gel gap, make the red blood cell of no aggegation have the ability of passing through the gel particle gap fully, the red blood cell of aggegation then can not pass through.
4, the present invention has superior protective system, select the organism benzoates as antiseptic, adopt methyl p-hydroxybenzoate and the propylparaben synergy (difference of the side carbochain length in the various P-hydroxybenzoic acid monoesters, thereby it is in the ability difference of permeates cell membranes, and its antibacterial action site is also just different, so various monoesters is just different at the inhibition ability of different types of microorganisms.Several esters compound has better antiseptic power.A large amount of practical application has both at home and abroad also confirmed the good antimicrobial effect of compound p-hydroxybenzoate than single p-hydroxybenzoate), effectively prevent the procreation of bacterium, obtain the storage life of long period, not only avoid using the Sodium azide chemical preservative that thereby the ionic strength of gel suspending medium system is promoted the sensitivity of ABO/RhD blood typing detection reagent card and the harmful effect of specificity generation simultaneously, also avoid using of the harmful effect of the antibiotics intermediate that institute's metabolism produces in the preservation process the specificity generation of ABO/RhD blood typing detection reagent card.
Another aspect of the present invention; in order to guarantee the quality of ABO/RhD blood typing detection reagent card of the present invention; in preparation process, also need gel is screened; promptly; at first to select suitable gel; generally the condition that should satisfy is: selecting particle diameter is the 30-60 nanometer, through the sephadex after the propylene acidylate.The gel raw material that screening is obtained need carry out swelling, washing, suspension, purpose is to allow the abundant swelling of gel particle, and gel particle, internal diameter super large or extra small gel particle and gel other impurity component in addition beyond the 30-60 nanometer of damaged gel particle, gathering removed in washing.After washing is finished with the gel suspending medium gel that suspends.
One side more of the present invention in order to guarantee the quality of ABO/RhD blood typing detection reagent card of the present invention, also needs the antibody raw material that mixes with gel is screened in preparation process.
In sum, the storage life why ABO/RhD blood typing detection reagent card of the present invention can have good specificity, sensitivity and reach 1 year, it is the synergy that is the various compositions of whole system, being arranged in of various gels can guarantee only to use a card just can finish positive reverse type of abo blood group and RhD blood typing on the card, buffer system can be kept the pH that the typing card reaction system needs; The low salt concn system can guarantee that gel particle obtains abundant swelling and gel particle diameter in needed scope.Lubricating system can guarantee lubricating ability suitable between the gel particle.Ester class antiseptic can prevent that gel or antibody lost efficacy because of bacterial reproduction.The gel of propylene acidylate can guarantee the suitable gap between the gel particle.Standardized antibody can ensure effectively detecting of antigen.
Detect abo blood group and RhD blood group with this ABO/RhD blood typing detection reagent card, the positive reaction of generation all is not less than 3+.Generally preserving the term of validity under 18-25 ℃ of condition was not less than 12 months.
In a word, enforcement of the present invention provides standardized ABO/RhD blood typing detection reagent card product, each hospital and Blood Transfusion Services can directly obtain the ABO/RhD blood typing detection reagent card of conformance to standard from the production supplier, for accurately carrying out positive reverse type of abo blood group and RhD blood typing, guaranteeing that safe transfusion created condition.
(4) embodiment
Below describe enforcement of the present invention and the beneficial effect that had in detail by specific embodiment, be intended to help the reader better to understand spirit of the present invention and essence, can not constitute any qualification to practical range of the present invention.
Embodiment 1:
The preparation of step 1, gel suspending medium
Described gel suspending medium prescription is as follows:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤4%,
Above reagent dissolved in distilled water, adjust pH are 6.6-6.8.
The preparation of step 2, gel
Select the Sephacryl gel for use, grain size is the 30-60 nanometer.
Wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation, remove the gel particle of damaged fragment of gel and gathering, collect and obtain even particle size and the complete gel that is suitable for spherical in shape.
The selection of step 3, antibody
Select the monoclonal anti-A antibody of IgM character, tire 〉=128
Select the monoclonal anti-B antibody of IgM character, tire 〉=128
Select the monoclonal anti-D of IgM character, tire 〉=64.
The preparation of step 4, gel
The gel of step 2 preparation is all mixed according to 2: 1～6: 1 ratio of volume ratio with each antibody of step 3 selection, be mixed with respectively the monoclonal anti-A antibody that contains IgM character gel, contain IgM character the monoclonal anti-B antibody gel and contain the gel of the monoclonal anti-D of IgM character; The gel of step 2 preparation is mixed according to 2: 1～6: 1 ratio of volume ratio with the gel suspending medium of step 1 preparation, be mixed with the gel that contains the gel suspending medium.
Step 5, packing
According to the amount of every pipe 22～28 microlitres, each gel that step 4 is prepared joins in six microtrabeculae pipes of a blank card, forms the ABO/RhD blood typing detection reagent card with 6 micro-column gel pipes.
Step 6, semi-manufacture are measured
Requirement contains in the micro-column gel pipe of antibody, has the positive reaction with the red blood cell generation 〉=3+ of antibody corresponding antigens, and promptly red blood cell concentrates on the gel upper surface, presents linear pattern.And produce negative reaction with the red blood cell that does not contain the corresponding antigen of antibody, promptly red blood cell can all arrive the microtubule bottom by gel, is deposited on the microtubule bottom.Red blood cell can all arrive the microtubule bottom by gel in the gel tube that antibody only contains gel suspending medium and gel mixture and do not contain, and be deposited on the microtubule bottom, presents negative reaction.
Step 7, seal
Pass through the press mold mode with the sealing suitable for reading of microtrabeculae pipe with aluminium-foil paper.Label behind the mark in 18-25 ℃ of preservation.
Step 8, preservation test
Above-mentioned ABO/RhD blood typing detection reagent card was preserved more than 1 year, and between this storage life, this ABO/RhD blood typing detection reagent card has following testing result:
(1) outward appearance:
Anti-A gel in the ABO/RhD blood typing detection reagent card is even blueness, anti-B gel is even yellow, the gel that anti-D gel and gel suspending medium suspend is even milky, and there is the as clear as crystal liquid of 1～2mm glue face upper end, and bubble and foreign matter do not have between the gel particle.
(2) sensitivity:
Contain in the micro-column gel pipe of antibody, have the positive reaction with the red blood cell generation 〉=3+ of antibody corresponding antigens, promptly red blood cell concentrates on the gel upper surface, presents linear pattern.
(3) specificity:
Contain in the micro-column gel pipe of antibody, have with the red blood cell of antibody corresponding antigens and produce positive reaction, promptly red blood cell concentrates on the gel upper surface, presents linear pattern.Produce negative reaction with the red blood cell that does not contain the corresponding antigen of antibody, promptly red blood cell can all arrive the microtubule bottom by gel, is deposited on the microtubule bottom.Red blood cell can all arrive the microtubule bottom by gel in the gel tube that antibody only contains gel suspending medium and gel mixture and do not contain, and be deposited on the microtubule bottom, presents negative reaction.
Embodiment 2:
Described ABO/RhD blood typing detection reagent card using method is as follows:
1, the ABO/RhD blood typing detection reagent card has six gel tubes.Wherein first pipe is anti-A gel, and second pipe is anti-B gel, and the 3rd pipe is anti-D gel, and the 4th to the 6th pipe is blank gel (gel that contains the gel suspending medium).Four, the 5th pipe adds serum to be checked (slurry) A type respectively, the Type B reagent red blood cell detects.The negative control tube of the 6th pipe.
2, with LISS solution or physiological saline person's red blood cell to be checked is mixed with 0.8～1% (percentage by weight) concentration, adds respectively in first, second, third and the 6th microtubule, every pipe 50 μ l.
3, known A type, Type B red blood cell are adjusted to 0.8～1% (percentage by weight) concentration with LI SS solution or physiological saline, join respectively in fourth, fifth microtubule, every pipe 50 μ l.
4, person's serum to be checked (slurry) is added in the the 4th, the 5th, the 6th microtubule every pipe 25 μ l respectively.
5, centrifugal behind the mixing with micro-column gel card special centrifugal machine, 900rpm 2 minutes, 1500rpm 3 minutes takes out back naked eyes result of determination, record.
6, result of determination
Positive findings: red blood cell floats in gel surface or the glue, positive reaction.
Negative findings: red blood cell is sunken to the bottom of micro-column gel.
The blood group judgement sees the following form:
| Anti-A | Anti-B | Anti-D | The A cell | The B cell | Negative control | The result |
| ??+ | ??0 | ??+ | ??0 | ??+ | ??0 | The A type D positive |
| ??0 | ??+ | ??+ | ??+ | ??0 | ??0 | The Type B D positive |
| ??0 | ??0 | ??+ | ??+ | ??+ | ??0 | The O type D positive |
| ??+ | ??+ | ??+ | ??0 | ??0 | ??0 | The AB type D positive |
| ??+ | ??0 | ??0 | ??0 | ??+ | ??0 | A type D feminine gender |
| ??0 | ??+ | ??0 | ??+ | ??0 | ??0 | Type B D feminine gender |
| ??0 | ??0 | ??0 | ??+ | ??+ | ??0 | 0 type D feminine gender |
| ??+ | ??+ | ??0 | ??0 | ??0 | ??0 | AB type D feminine gender |
Annotate: "+" positive " 0 " is negative
Claims (1)
1. the preparation method of an ABO/RhD blood typing detection reagent card, have 6 micro-column gel pipes on the described reagent card, the gel tube that contains the gel suspending medium that the gel tube of the gel tube of the monoclonal anti-B antibody that described 6 micro-column gel pipes are respectively the pipe gel tubes that contain the monoclonal anti-A antibody of IgM character, a pipe contains IgM character, the monoclonal anti-D that a pipe contains IgM character, the gel tube that contains the gel suspending medium that two pipes are used for reverse type and a pipe are used for negative control, it is characterized in that: described method comprises following technological process:
The preparation of step 1, gel suspending medium
Described gel suspending medium prescription is as follows:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤4%,
Above reagent dissolved in distilled water, adjust pH are 6.6-6.8,
The preparation of step 2, gel
Select the Sephacryl gel for use, grain size is the 30-60 nanometer, wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation, remove the gel particle of damaged fragment of gel and gathering, collection obtains gel spherical in shape even particle size and complete
The selection of step 3, antibody
Select the monoclonal anti-A antibody of IgM character, tire 〉=128
Select the monoclonal anti-B antibody of IgM character, tire 〉=128
Select the monoclonal anti-D of IgM character, tire 〉=64,
The preparation of step 4, gel
The gel of step 2 preparation is all mixed according to 2: 1～6: 1 ratio of volume ratio with each antibody of step 3 selection, be mixed with respectively the monoclonal anti-A antibody that contains IgM character gel, contain IgM character the monoclonal anti-B antibody gel and contain the gel of the monoclonal anti-D of IgM character; The gel of step 2 preparation is mixed according to 2: 1～6: 1 ratio of volume ratio with the gel suspending medium of step 1 preparation, is mixed with the gel that contains the gel suspending medium,
Step 5, packing
According to the amount of every pipe 22～28 microlitres, each gel that step 4 is prepared joins respectively in six microtrabeculae pipes of a blank card, forms the ABO/RhD blood typing detection reagent card with 6 micro-column gel pipes.
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|---|---|---|---|
| CN 200910234608 CN101718794B (en) | 2009-11-25 | 2009-11-25 | Preparation method of ABO/RhD blood typing detection reagent card |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910234608 CN101718794B (en) | 2009-11-25 | 2009-11-25 | Preparation method of ABO/RhD blood typing detection reagent card |
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| CN101718794B CN101718794B (en) | 2013-06-05 |
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